TransMessenger Transfection Reagent
For transfection of cells with RNA
Features and benefits
- Efficient transfection of RNA
- Quick and easy setup
- Ready-to-use reagent
- Tested for absence of RNase activity
Principle
TransMessenger Transfection Reagent
is the first reagent specifically developed for transfection of cells with RNA.
TransMessenger Reagent provides a fast and easy procedure and high transfection
efficiencies (see figure "TransMessenger Reagent with HeLa
Cells"). The reagent is a lipid-based formulation that is used in
conjunction with a specific RNA-condensing enhancer and an optimized buffer. RNA
molecules are condensed by the enhancer and then coated by TransMessenger
Reagent for efficient transfer into eukaryotic cells. Strict quality control is
performed to test for absence of RNase activity, lot-to-lot consistency, and low
endotoxin levels (≤10 EU/ml). Our rigorous standards eliminate reagent
variables that can adversely affect the efficiency of RNA transfection.
TransMessenger
Reagent with HeLa Cells
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Expression of green fluorescence
protein (GFP) in HeLa-S3 cells. 8 x 104 cells
were seeded into 48-well plates and transfected 24 h later with 0.5 µg
of an in vitro-transcribed GFP-encoding RNA (transcribed from
P7ASP-GFP/Mlu) using 1 µl Enhancer R and 2.5 µl
TransMessenger Reagent. Cells were analyzed 24 h post-transfection
by fluorescence microscopy. Approximately 50% of the cells were
successfully transfected. (P7ASP-GFP/Mlu kindly provided by J.
Bogenberger, Stanford University Blood Center, Palo Alto, CA, USA.)
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Procedure
All TransMessenger Reagent components
are provided as ready-to-use solutions. To generate TransMessenger–RNA
transfection complexes, simply mix your RNA with Enhancer R and Buffer EC-R and
incubate for 5 minutes at room temperature, then add TransMessenger Reagent
and incubate for a further 5–10 minutes. The complexes are mixed with
medium and added directly to the cells. Following a 3 hour incubation, the
medium is changed and the cells are incubated until they are ready for analysis.
Optimal transfection results are achieved using high-purity RNA that is free of
DNA, proteins, and other contaminants. RNA purified with RNeasy and Oligotex
mRNA Kits is highly recommended. Since the amount of RNA is a critical factor
for successful transfection, we recommend optimizing the amounts of RNA and
TransMessenger Transfection Reagent for every cell type–RNA combination (see
figure "Amount of RNA and TransMessenger Reagent vs.
Transfection Efficiency"). To facilitate this, the reagent is provided
with guidelines for optimization and starting points for optimization in
different cell-culture formats.
Amount of RNA and
TransMessenger Reagent vs. Transfection Efficiency
Optimization experiments in CHO-K1 cells.
2 x 104 cells were seeded in quadruplicate
into 96-well plates and transfected 24 h later with an in vitro-transcribed
CAT-encoding RNA using A increasing
amounts of RNA with 1.5 µl TransMessenger Reagent and B increasing
amounts of TransMessenger Reagent with 0.25 µg RNA, as described in
the TransMessenger Transfection Reagent Handbook.
CAT activity was measured 24 h post-transfection.
Applications
Transfection of cells with RNA rather
than DNA provides an alternative approach that offers new possibilities for
transfection experiments. For example, RNA transfection could be useful for
studying cells that are not efficiently transfected with plasmid DNA, and could
allow direct studies of RNA function. Transfected RNA sequences are expressed in
the absence of transcription and in a promoter-independent manner. In addition,
protein expression usually occurs sooner following transfection of RNA than
following transfection of DNA.